EP0198386A2 - Anhaltende Hochproduktion von menschlichem rekombinantem gamma-Interferon unter Verwendung eines Rinderpapillomavirus-Vektors - Google Patents

Anhaltende Hochproduktion von menschlichem rekombinantem gamma-Interferon unter Verwendung eines Rinderpapillomavirus-Vektors Download PDF

Info

Publication number
EP0198386A2
EP0198386A2 EP86104742A EP86104742A EP0198386A2 EP 0198386 A2 EP0198386 A2 EP 0198386A2 EP 86104742 A EP86104742 A EP 86104742A EP 86104742 A EP86104742 A EP 86104742A EP 0198386 A2 EP0198386 A2 EP 0198386A2
Authority
EP
European Patent Office
Prior art keywords
vector
host
interferon
vector according
expression
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP86104742A
Other languages
English (en)
French (fr)
Other versions
EP0198386A3 (de
Inventor
Nava Sarver
George A. Ricca
William N. Drohan
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Meloy Laboratories Inc
Original Assignee
Meloy Laboratories Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Meloy Laboratories Inc filed Critical Meloy Laboratories Inc
Publication of EP0198386A2 publication Critical patent/EP0198386A2/de
Publication of EP0198386A3 publication Critical patent/EP0198386A3/de
Withdrawn legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • C07K14/57—IFN-gamma
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20041—Use of virus, viral particle or viral elements as a vector
    • C12N2710/20043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
    • C12N2830/002—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/30—Vector systems having a special element relevant for transcription being an enhancer not forming part of the promoter region
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/80—Vector systems having a special element relevant for transcription from vertebrates
    • C12N2830/85—Vector systems having a special element relevant for transcription from vertebrates mammalian

Definitions

  • This invention relates to a process for producing recombinant human immune interferon. More particularly, the invention relates to a process for production of recombinant human immune interferon in mammalian cell expression systems using a bovine papillomavirus shuttle vector.
  • Interferon is a glycoprotein whose synthesis in cells is principally induced by viruses or mitogens. Interferons are classified into three major species designated IFN- « (leukocyte), IFN-p (fibroblast), and IFN-y (immune). Interferons possess potent antiviral, anticellular, immunoregulatory, and antitumor activities. Interferon treatment of cancer and viral infections in animals has met with some success. Consequently, immune interferon is of great interest clinically as well as academically.
  • DNA deoxyribonucleic acid -
  • methods are currently in use for delivering defined foreign deoxyribonucleic acid - (DNA) segments into eukaryotic cells. These include physical injection of DNA [Mueller, C., A. Graessmann, and M. Graessmann, Cell (1978) 15:579], fusion of DNA containing liposomes - [Dimitriadis, G.J., Nature (London) (1978) 274 :923)] or erythrocytes [Rechsteiner, M., Natl. Cancer Inst. Monoar. (1978) 48 :57] with target cells, and the direct application of naked DNA onto cells in the presence of calcium phosphate [Graham, F.L., and A.J. van der Eb.
  • SV40 Session virus 40
  • a segment of the viral genome is covalently linked to defined nucleic acid segments [Gruss, P., and G. Khoury, Proc. Natl. Acad. Sci. (1981) 78:133; Hamer, D.H., and P. Leder, Nature (1979), 281:35-40; and R.C. Mulligan, et al., Nature (London) (1979) 277:108].
  • the SV40 vector system offers a rapid and efficient way to introduce foreign DNAs into permissive host cells, the system is limited by the size of DNA that can be accomodated within the virus particle.
  • SV40 DNA has not yet been exploited as a cloning vector in non- permissive rodent cells, because (i) SV40 transformation is associated with integration of the viral genome, a process that may disrupt the integrity of the foreign DNA segment of interest, and (ii) there is no indication that the gene will be active at detectable levels from the low integrated copy numbers which are sufficient for the expression of the SV40-transforming gene.
  • bovine papillomavirus (BPV) DNA as a viral vector is well documented and was originally based on the observation that the viral genome persists as an extrachromosomal plasmid in transformed cells (Law, M.-F., D.R. Lowy, I. Dvoretzky, and P.M. Howley, Proc. Natl. Acad. Sci., USA 78:2727:2731, 1981).
  • the cloned gene is maintained in a uniform sequence environment of the BPV minichromosome eliminating potential problems associated with the integration of the cloned DNA into inactive regions of the host chromosome.
  • Another improvement in the BPV vector system was the incorporation of a dominant selectable marker which has expanded the range of cells capable of supporting amplified BPV plasmid replication beyond those manifesting the transformed phenotype.
  • This invention realizes the utility of BPV DNA to imortalize cells and to exist as multicopy plasmid in transduced cells. It further describes adaptation of these transduced cells for prolonged amplified expression of a foreign gene using human gamma IFN cDNA as the model gene.
  • the present invention relates to a bovine papillomavirus expression vector capable of replication in a prokaryotic host and replication and expression of. non-native genetic information in an eukaryotic host comprising a prokaryotic promoter, a eukaryotic promoter,, at least two dominant selectable markers, a signal sequence, and an enhancer element.
  • the present invention has made it possible to provide readily available large quantities of human immune interferon. This has been achieved by the application of recombinant DNA technology to preparing cloning vehicles encoding for the human immune interferon protein, and by screening/ isolating procedures for recovering human immune interferon essentially free of other proteins of human origin.
  • the present invention provides human immune interferon or its fragments essentially free of other proteins of human origin.
  • the immune interferon protein is glycosylated.
  • Immune interferon is produced by recombinant DNA techniques in host cells or other self-replicating systems and is provided in essentially pure form. Also provided are methods and compositions for preparing the above-described interferon as well as therapeutic compositions and uses for the interferon protein or fragments in the treatment of cancer or viral infections in humans and animals.
  • the invention further provides replicable expression vectors incorporating a DNA sequence encoding immune interferon and a self-replicating host cell system transformed or transfected thereby.
  • the host system is mammalian cells.
  • the human immune interferon is produced by a process which comprises (a) preparing a replicable expression vector capable of expressing the DNA sequence encoding interferon in a suitable host cell system; (b) transforming said host system to obtain a recombinant host system; (c) maintaining said recombinant host system under conditions permitting expression of said interferon encoding DNA sequence to produce immune interferon protein; and (d) recovering said interferon protein.
  • the interferon-encoding replicable expression vector is made by preparing a double-stranded complementary DNA (ds-cDNA) preparation representative of interferon mRNA and incorporating the ds-cDNA into replicable expression vectors within a eukaryotic transcription cassette containing regulatory elements required for transcription of said cDNA.
  • the preferred mode of recovering the interferon comprises reacting the proteins expressed by the recombinant host system with a reagent composition comprising at least one binding protein specific for interferon. Binding proteins such as antibodies to IFN-y, either monoclonal or polyclonal, would be useful in the recovery step. See for example the antibody described by Lee J., et al. (IL lmmunol. Methods 69(1) 61-70 (1984)).
  • the invention provides a bovine papillomavirus expression vector capable of dual host replication by virtue of the presence of a prokaryotic replicon and a eukaryotic replicon.
  • the vector also contains a eukaryotic promoter, a prokaryotic promoter, at least two dominant selectable markers-and., a signal sequence.
  • this invention provides various derivatives of BPV expression . vectors characterized in possessing increased expression capabilities when compared to a standard vector.
  • the invention provides a host transformed by a bovine papillomavirus expression vector capable of replication in a prokaryotic replicon, a eukaryotic replicon, a eukaryotic promoter, at least two dominant selectable markers, and a signal sequence.
  • the invention provides a process for producing human interferon comprising providing a replicable expression vector capable of expressing the DNA sequence encoding immune interferon in a suitable host, transforming said host, and maintaining said transformed host under conditions permitting the expression of said interferon, the improvement which comprises the employing the vector pdBV-y -IFN as the replicable expression vector.
  • Figure 1 illustrates the physical map of pdBPV- human gamma interferon (p8-4) DNA (top). The human gamma interferon cDNA insert is shown in greater detail (bottom).
  • the plasmid consists of the following structural elements described in a clockwise direction.
  • the complete BPV genome is joined to the pML2dl sequence containing the pBR322 origin of replication and a functional beta-lactamase gene.
  • the moloney murine sarcoma virus enhancer (MSV) was originally derived from the proviral LTR as a Hinfl-Xbal fragment (141-525, respectively). Both termini had previously been converted to BamH1 sites with synthetic linkers.
  • the mouse metallothionein I promoter is a Kpnl to Bg1II fragment of approximately 600 bp. The Kpnl site was previously modified to a Bg1II site to facilitate cloning.
  • the human gamma-interferon cDNA Downstream from the promoter is the human gamma-interferon cDNA obtained as a Sau3A fragment end is followed by a non-functional 3' region of the neomycin gene.
  • the 3' processing signals are contained within an 850 bp fragment derived from SV40 DNA.
  • a Sau3A fragment (843 bp) consists of the complete coding sequence for human gamma-interferon including the signal peptide and is shown in detail at the bottom of Figure 1.
  • the 5' and 3' untranslated regions contain 60 bp and 286 bp, respectively.
  • Figures 3A and 2B illustrate the expression levels of recombinant ⁇ -IFN in isolated foci and in single cell clones of transformed C127 cells.
  • DNA transfections were performed using the calcium phosphate coprecipitation method followed by enhancement with 25% dimethylsulfoxide as described previously. Phenotypically transformed foci were isolated and established as cell lines. Single cell cloning was achieved by plating at limited dilution into 96 well plates, visual identification of wells containing a single cell, and propagation of these cells into established lines.
  • y-IFN activity was assayed by a cytopathic effect inhibition assay using 3-fold serial dilutions of samples on human WISH cells challan- ged with encephalomyocarditis virus (EMC).
  • EMC encephalomyocarditis virus
  • Figure 3 illustrates the requirement for fetal calf serum in long-term expression of recombinant y-IFN.
  • the production of ⁇ -IFN was examined as a function of the serum concentration in the medium.
  • Duplicate cultures of transformed cells in 60 mm dishes were grown in the presence or absence of FCS, and the culture media was harvested daily and assayed for the presence of biologically active -y-IFN.
  • Figure 4 illustrates the physical structure of the SV40 and neomycin genetic elements.
  • Figure 5 illustrates the physical structure of BPV/gamma-interferon vectors with modified 3' ends.
  • Four vectors were derived from the same parental plasmid containing the following elements (described in a clockwise direction): the complete BPV genome; pML2d1; Maloney murine sarcoma virus enhancer (MSV EH ); metallothionein promoter - (MT PRO ); and human gamma-interferon cDNA.#The constructs differ in the sequences 3' to the gamma-interferon cDNA;
  • FIG. 6 illustrates the physical map of pdBPV- human gamma-interferon (p72-4) DNA.
  • p72-4 is a modified BPV/gamma-interferon plasmid derived from p8-4 after deleting the Neo-3' fragment (314 bp).
  • the plasmid consists of the following structural elements described in a clockwise direction: the complete BPV genome is joined to the pML2d1 sequence containing the pBR322 origin of replication and a functional beta-lactamase gene.
  • the Maloney murine sarcoma virus enhancer (MSV) was originally derived from the proviral LTR as a Hinfl-Xbal fragment (141-525, respectively).
  • the mouse metallothionein I promoter is a KDnl to Bg1 II fragment of approximately 600 bp.
  • the K D nl site was previously modified to a Bg1II site to facilitate cloning.
  • Downstream from the promoter is the human gamma-interferon cDNA obtained as a Sau3A fragment which is followed by RNA processing signals derived from SV40 DNA.
  • Figure 7 illustrates the immunoprecipitation of ex t racullular ⁇ -IFN secreted from NS8-4-G-4-transformants.
  • Cells in 100 mm plates were washed with methionine-free medium (GIBCO) containing 2% dialyzed fetal calf serum (FCS) and labeled in 3 ml of the same medium with 400 uCi/ml of 1- 35 -S-methionine (1063.6 Ci/mmol; New England Nuclear) for 4 hours at 37°C. with anti-human gamma interferon serum as described previously.
  • GEBCO methionine-free medium
  • FCS fetal calf serum
  • the antiserum was first preincubated with 2 ug of human y-IFN after which the complex was added was performed on a 10% SDS-polyacrylamide gel after which the gels were treated with En 3Hance (New England Nuclear) and fluorographed.
  • Lane C Goat anti-y-IFN serum neutralized with 2 uf of y-IFN prior to immunoprecipitation.
  • Lane D Extracellular medium is from transformants harboring a recombinant DNA in which the y-IFN cDNA is in a transcriptional direction opposite that of the metallathione in promoter. The sample was treated with goat anti-y-IFN serum.
  • interferon denotes human immune interferon or its fragments produced by cell or cell-free culture systems, in bioactive forms having the capacity to influence cellular growth and viral replication as does immune interferon native to the human plasma.
  • interferon Different alleles of interferon may exist in nature. These variations may be characterized by difference(s) in the nucleotide sequence of the structural gene coding for proteins of identical biological function. In addition, the location and degree of glycosylation as well as other post-translation modifications may vary and will depend to a degree upon the nature of the host and environment in which the protein is produced. It is possible to produce analogs having single or multiple amino acid substitutions, deletions, additions, or replacements. All such allelic variations, modifications, and analogs resulting in derivatives of interferon which retain the biologically active properties of native interferon are included within the scope of this - invention.
  • Expression vectors refer to vectors which are capable of transcribing and translating DNA sequences contained therein, where such sequences are linked to other regulatory sequences capable of effecting their expression. These expression vectors must be replicable in the host organisms or systems either as episomes, bacteriophage, or as an integral part of the chromosomal DNA.
  • One form of expression vector which is particularly suitable for use in the invention is bovine papillomavirus (PBV) DNA.
  • shuttle vector means the vector contains the necessary replication regions - (replicons) to allow replication in two distinct hosts.
  • the shuttle vectors of this invention are capable of replication in E. coli, a prokaryotic organism due to the presence of a bacterial replicon contained in the plasmid PML2D1, itself a derivative of plasmid pBR322 as well as capable of replication in an PBV susceptible eukaryotic cell line due to the BPV replicon.
  • Bovine papillomavirus 1 DNA as well as a cloned 69% subgenomic fragment of the bovine papillomavirus 1 genome are very efficient in inducing transformed foci in susceptible mouse cell [Howley, P.M., et al. 1980, in M. Essex, et al., (ed.), Viruses in Naturally Occurring Cancers, (Cold Spring Harbor Laboratory; Cold Spring Harbor, New York) p. 233-247 and Lowry, D.R., et al., Nature (London) (1980) 287:72].
  • Bovine papillomavirus-transformed cells contain multiple copies (10 to 120 per cell) of the viral DNA.
  • Recombinant vectors and methodology disclosed herein are suitable for use in host cells covering a wide range of prokaryotic and eukaryotic organisms.
  • pro- kayotes are preferred for the cioning and amplification of DNA sequences and in the construction of vectors.
  • E. coli K12 strain HB101 - ATCC No. 33694
  • other microbial strains may be used.
  • Vectors containing replication and control sequences which are derived from species compatible with the host cell or system are used in connection with these hosts.
  • the vector ordinarily carries an origin of replication, as well as-genetic characteristics capable of providing phenotypic selection in transformed cells.
  • E. coli can be transformed using the vector pBR322, which contains the promoters and genes for ampicillin and tetracycline resistance - [Bolivar, et al., Gene, 2:94 (1977)].
  • the expression vector may also contain control elements which can be used by the vector for expression of its own proteins.
  • Common prokaryotic control elements used for expression of foreign DNA sequences in E. coli include the promoters and regulatory sequences derived from the 8-galactosidase and tryptophan (trp) operons of E. coli, as well as the pR and pL promoters of bacteriophage lambda. Combinations of these elements have also been used (e.g., TAC, which is a fusion of the trp promoter with the lactose operator).
  • Other promoters have also been discovered and utilized, and details concerning their nucleotide sequences have been published enabling a skilled worker to combine and exploit them functionally.
  • cultures of cells derived from multicellular organisms may also be used as hosts.
  • any such cell culture is workable, whether from a vertebrate or invertebrate source.
  • interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure in recent years.
  • useful hosts are the mouse L cells, VERO, HeLa, mouse C127, Chinese hamster ovary (CHO), W138, BHK, COS-7, and MDBK cell lines.
  • Expression vectors for such cells ordinarily include an origin of replication, a promoter located upstream of the gene to be expressed, along with any RNA splice sites, polyadenylation site, and transcription termination sequence.
  • control functions on the expression vectors are often provided by viral derived sequences.
  • promoters are derived from polyoma, Adenovirus 2, and most frequently, Simian Virus 40 (SV40).
  • SV40 Simian Virus 40
  • eukaryotic enhancer sequences can also be added to the construction. These sequences can be obtained from a variety of DNA viruses such as SV40, polyoma or oncogenic retroviruses such as the mouse sarcoma virus and mammary tumor virus.
  • a mammalian origin of replication is usually part of the viral vector used as a vehicle, such as that provided by SV40 or other viral sources, or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter is often sufficient.
  • Host cells can prepare interferon proteins which can be a variety of chemical compositions.
  • the protein is produced having methionine as its first amino acid (present by virtue of the ATG start signal codon naturally existing at the beginning of the structural gene or inserted before a segment of the structural gene).
  • the protein may also be intra-or extracellularly cleaved, giving rise to the amino acid which is found naturally at the amino terminus of the protein.
  • the protein may be produced together with either its signal polypeptide or a conjugated protein other than the convention signal polypeptide, the signal polypeptide of the conjugate being specifically cleavable in an intra or extracellular environment.
  • interferon may be produced by direct expression in mature form without the necessity of cleaving away any extraneous polypeptide.
  • the signal sequence used herein is the y-IFN signal sequence described by Devos, et al. - (Nucleic Acid Res. 10:2487-1501 (1982) and Gray, et al. (Nature 295:503-508 (1982)) which includes the coding region for an additional three amino acids (lys, tyr, cys) 3' to the region reported.
  • This modified sequence is a unique signal sequence construction and is useful for the expression and export of a variety of other protein products in addition to IFN-y for which open reading found sequences are available.
  • Recombinant host cells refer to cells which have been transformed with vectors constructed using recombinant DNA techniques. As defined herein, immune interferon is produced as a consequence of this transformation. Immune interferon or its fragments produced by such cells are referred-to as "recombinant immune interferon”.
  • Double-stranded cDNA (ds-cDNA) is in-' serted into the expression vector by any one of many known techniques. In general, methods, etc., can be found in Maniatis, supra, and Methods In Enzymology, Volumes 65 and 68 (1980); and Volume 100 and 101 (1983). In general, the vector is linearized by at least one restriction endonuclease, which will produce at least two blunt or cohesive ends.
  • the ds-cDNA is ligated with or joined to the vector insertion site.
  • prokayotic cells or other cells which contain substantial cell wall material are employed, the most common method of transformation with the expression vector is calcium chloride pretreatment as described by Cohen, R.N., et al., Proc. Natl. Acad. Sci. USA, 69:2110 (1972). If cells without cell wall barriers are used as host cells, transfection is carried out by the calcium phosphate coprecipitation method described by Graham and Van der Eb, Virology, 52:456 (1973). Other methods for introducing DNA into cells such as nuclear injection or protoplast fusion, have also been successfully used. The organisms are then cultured on selective media and proteins for which the expression vector encodes are produced.
  • Clones containing the entire gene for immune interferon are identified in a semi-micro C.P.E. inhibition assay using WISH cells as the indicator cells and EMC as the challenge virus.
  • This method of identification requires that the ds-cDNA be inserted into a vector containing appropriate regulatory nucleic acid sequences adjacent to the insertion site. These regulatory sequences initiate transcription and translation of those ds-cDNA molecules inserted in the vector. Those clones containing interferon cDNA sequences correctly positioned relative to the regulatory sequences synthesize part or all of the interferon protein. Restriction endonuclease digestion and nucleotide sequencing techniques are used to determine the sequence of amino acids encoded by the cDNA fragments.
  • the gamma interferon cDNA clone employed herein was obtained as follows. Briefly, size fractionated mRNA was isolated from human peripheral blood lymphocytes that had been induced with A23187 and mezzarine (I.A. Braude, Biochemistrv, 23:5603, 1984). The mRNA was converted into double-stranded cDNA and cloned into the Pstl site of pBR322 by G-C tailing using standard procedures. An 843 base pair Sau3A fragment containing the gamma interferon signal peptide, coding region, and translation termination codon, was cloned into a BPV derivative vector as shown in Figure 1.
  • the resulting construct contained gamma interferon sequences placed downstream from a mouse metallothienein 1 promoter, MSV enhancer sequence, and also contained the SV40 small t splice sites and polyadenylation site.
  • the vector also contains 314 b.p. between the 3' end of the y-IFN cDNA and the SV40 elements.
  • the 314 b.p. are derived from the 3' ends of the neomycin gene (See Figure 4B).
  • C127 mouse cells were transformed using the calcium phosphate coprecipitation method (Graham and van der Eb, supra) followed by enhancement with 25% dimethylsulfoxide (Stowe and Wilkie, J. Gen. Virol., 33:447, 1976) as described previously - (Sarver, et al., Mol. Cell Biol., 1:486, 1981). Phenotypically transformed foci were isolated and established as cell lines. Single cell cloning was achieved by plating at limited dilution into 96 well plates (Co-star), incubating at 37°C., identifying wells containing a single cell, and propagating these cells into established lines.
  • tissue culture media from transformed cells derived from 16 individual foci was collected and assayed for the presence of biologically active gamma interferon.
  • the biological assay specifically measured a reduction in the cytopathic effect of EMC on human WISH cells.
  • the results in Figure 2A demonstrate a large variation in the amount of biologically active gamma interferon produced by different foci.
  • the analysis was performed on 24 independent single-cell clones obtained from a single transformed focus (number 8-4-G), the amount of biologically active gamma interferon produced differed over a much smaller range (Figure 2B).
  • BPV transformed cells Having identified gamma interferon biological activity BPV transformed cells, the nature of the polypeptide product was investigated.
  • Cells were labelled with 35 S-methionine, extracellular media was harvested and immunoprecipitated with a polyclonal goat anti-gamma interferon serum. The precipitated proteins were then analyzed on SDS polyacrylamide gels as described elsewhere - (Sarver, et al., Mol. Cell. Biol. 1:486-496 (1981)).
  • SDS polyacrylamide gels as described elsewhere - (Sarver, et al., Mol. Cell. Biol. 1:486-496 (1981)).
  • Analysis of BPV transformed cells demonstrated the presence of a prominent band identical in its electrophoretic mobility to native gamma interferon (Figure 7). This band was absent from the media obtained from cells transformed by BPV constructs containing gamma interferon cDNA inserted in the opposite orientation.
  • the production of crude gamma interferon was subsequently examined as a function of the serum concentration in the medium.
  • Duplicate cultures of transformed cells were grown in the presence or absence of fetal calf serum.
  • the culture media was harvested daily and assayed for the presence of biologically active gamma interferon.
  • the data in Figure 3 demonstrated that approximately 10-fold more gamma interferon is produced in cells maintained in the presence of 2.5% fetal calf serum when compared with cells maintained in the absence of serum.
  • the recombinant protein is biologically indistinguishable from nature ⁇ -IFN in that it confers antiviral protection on appropriate host cells, induces the expression of class II major histocompatibility - (MHC) antigens in HeLa and endothelial cells and is neutralized by anti-human--y-IFN serum.
  • MHC major histocompatibility -
  • Its identity with authentic human y-IFN is further illustrated by the fact that a monoclonal antibody (provided by Rebesh Rubin, New York Blood Center) which recognized nature y-IFN isolated from included peripheral blood leukocytes, but not bacterially produced recombinant human -y-IFN, completely neutralizes the y-IFN obtained from the engineered C127 cells.
  • the region of modification comprises an inactive fragment of the neomycin resistance gene, a region of SV40 DNA containing 1) small t-splice sites which have been implicated in the post-transcriptional modification of mRNAs, as well as 2) a polyadenylation site.
  • posttranscriptional regulatory includes sequences involved with mRNA processing events such as the small t-splice regions and poly A addition sequences described above.
  • SV40 small "t" intron has an augmenting effect on gamma interferon cDNA expression.
  • p70-21, deleted of SV40 splicing signals is only 50% as efficient is splicing gamma interferon as p8-4, the original vector.
  • the cell lines have been in culture for at least four months and are stable with regard to the level of human gamma interferon expression.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Physics & Mathematics (AREA)
  • Toxicology (AREA)
  • Virology (AREA)
  • Plant Pathology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
EP86104742A 1985-04-05 1986-04-07 Anhaltende Hochproduktion von menschlichem rekombinantem gamma-Interferon unter Verwendung eines Rinderpapillomavirus-Vektors Withdrawn EP0198386A3 (de)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US72009285A 1985-04-05 1985-04-05
US78374385A 1985-10-03 1985-10-03
US783743 1985-10-03
US720092 1996-09-27

Publications (2)

Publication Number Publication Date
EP0198386A2 true EP0198386A2 (de) 1986-10-22
EP0198386A3 EP0198386A3 (de) 1988-01-07

Family

ID=27110200

Family Applications (1)

Application Number Title Priority Date Filing Date
EP86104742A Withdrawn EP0198386A3 (de) 1985-04-05 1986-04-07 Anhaltende Hochproduktion von menschlichem rekombinantem gamma-Interferon unter Verwendung eines Rinderpapillomavirus-Vektors

Country Status (4)

Country Link
EP (1) EP0198386A3 (de)
AU (1) AU595509B2 (de)
CA (1) CA1305930C (de)
ES (1) ES8800988A1 (de)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS63141581A (ja) * 1986-12-05 1988-06-14 Toray Ind Inc 形質転換体および糖付加ポリペプチドの生産方法
US6384194B1 (en) 1987-12-16 2002-05-07 Dsm N.V. Expression and purification of human interleukin-3 and muteins thereof
US6767699B2 (en) 2000-05-31 2004-07-27 Chiron Corporation Method for the quantitation of alphavirus replicon particles

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4419446A (en) * 1980-12-31 1983-12-06 The United States Of America As Represented By The Department Of Health And Human Services Recombinant DNA process utilizing a papilloma virus DNA as a vector

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4419446A (en) * 1980-12-31 1983-12-06 The United States Of America As Represented By The Department Of Health And Human Services Recombinant DNA process utilizing a papilloma virus DNA as a vector

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
CHEMICAL ABSTRACTS, vol. 100, no. 15, 9th April 1984, page 138, abstract no. 115824x, Columbus, Ohio, US; G. MENEGUZZI et al.: "Plasmidial maintenance in rodent fibroblasts of a BPV1-pBR322 shuttle vector without immediately apparent oncogenic transformation of the recipient cells", & EMBO J. 1984, 3(2), 365-71 *
CHEMICAL ABSTRACTS, vol. 100, no. 17, 23rd April 1984, page 150, abstract no. 133436b, Columbus, Ohio, US; M. KRIEGLER et al.: "Promoter substitution and enhancer augmentation increases the penetrance of the SV40 A gene to levels comparable to that of the Harvey murine sarcoma virus ras gene in morphologic transformation", & UCLA SYMP. MOL. CELL. BIOL., NEW SER. 1983, 8(GENE EXPRESSION), 107-24 *
CHEMICAL ABSTRACTS, vol. 104, no. 19, 12th May 1986, page 156, abstract no. 162935b, Columbus, Ohio, US; N. SARVER et al.: "Sustained high-level expression of recombinant human gamma interferon using a bovine papillomavirus vector", & UCLA SYMP. MOL. CELL. BIOL., NEW SER. 1985, 32(PAPILLOMAVIRUSES), 515-27 *
CHEMICAL ABSTRACTS, vol. 97, no. 9, 30th August 1982, page 153, abstract no. 67176h, Columbus, Ohio, US; M.F. LAW et al.: "Expression of selective traits in mouse cells transformed with a BPV DNA-derived hybrid molecule containing Escherichia coli gpt.", & EUKARYOTIC VIRAL VECTORS, ÄCONF.Ü, 1981, (PUB. 1982), 79-85 *
CHEMICAL ABSTRACTS, vol. 98, no. 5, 31st January 1983, page 189, abstract no. 28920x, Columbus, Ohio, US; N. SARVER et al.: "Transformation and replication in mouse cells of a bovine papillomavirus-pML2 plasmid vector that can be rescued in bacteria", & PROC. NATL. ACAD. SCI. U.S.A. 1982, 79(23), 7147-51 *
EUKARYOTIC VIRAL VECTORS, 1982, conf. 1981, pages 87-92, Cold Spring Harbor Laboratory, GB; B. BINETRUY et al.: "Bovine papilloma virus 1-pBR322 and polyoma-pBR322 recombinants as eukaryotic vectors" *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCE OF THE USA, vol. 79, July 1982, pages 4030-4034; D. DIMAIO et al.: "Bovine papillomavirus vector that propagates as a plasmid in both mouse and bacterial cells" *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE USA, vol. 81, no. 16, August 1984, pages 5086-5090, Washington, US; R. FUKUNAGA et al.: "Constitutive production of human interferons by mouse cells with bovine papillomavirus as a vector" *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS63141581A (ja) * 1986-12-05 1988-06-14 Toray Ind Inc 形質転換体および糖付加ポリペプチドの生産方法
US6384194B1 (en) 1987-12-16 2002-05-07 Dsm N.V. Expression and purification of human interleukin-3 and muteins thereof
US6767699B2 (en) 2000-05-31 2004-07-27 Chiron Corporation Method for the quantitation of alphavirus replicon particles

Also Published As

Publication number Publication date
ES8800988A1 (es) 1987-12-16
CA1305930C (en) 1992-08-04
AU595509B2 (en) 1990-04-05
ES554184A0 (es) 1987-12-16
AU5576786A (en) 1986-10-09
EP0198386A3 (de) 1988-01-07

Similar Documents

Publication Publication Date Title
US4889803A (en) Production of interferon gamma
US4840896A (en) Heteropolymeric protein
US5554514A (en) Production of recombinant human interferon beta2.sbsb.B
EP0077670B1 (de) Humanes Immuninterferon
Soprano et al. Mutational analysis of simian virus 40 T antigen: stimulation of cellular DNA synthesis and activation of rRNA genes by mutants with deletions in the T-antigen gene
Haynes et al. Constitutive, long-term production of human interferons by hamster cells contalning multiple copies of a cloned interferon gene
JP2633227B2 (ja) 動物インターフエロン
EP0957167A1 (de) Herstellung heterodimerer menslicher Fruchtbarkeitshormone
US5582824A (en) Recombinant DES-CYS-TYR-CYS human immune interferon
EP0237019A2 (de) Konjugiertes Interferon und dessen Herstellung unter Verwendung eines rekombinanten Gens
EP0220574B2 (de) Menschliches Interferon-beta2A ; Vektoren, die Gene enthalten, die für das genannte Interferon kodieren; Zellinien, die dieses produzieren und Verwendung des genannten Interferons als Pharmazeutikum
EP0390252A2 (de) Reinigung von rekombinantem, menschlichem Interleukin-3
EP0517768A1 (de) Expressionsvektoren für säugetierzellen
Mory et al. Efficient constitutive production of human IFN-γ in Chinese hamster ovary cells
US4939088A (en) Sustained production of recombinant gamma interferon using an Epstein-Barr virus replicon
US5831023A (en) Recombinant animal interferon polypeptides
EP0198386A2 (de) Anhaltende Hochproduktion von menschlichem rekombinantem gamma-Interferon unter Verwendung eines Rinderpapillomavirus-Vektors
Pitha et al. Induction of human beta-interferon synthesis with poly (rI. rC) in mouse cells transfected with cloned cDNA plasmids.
US6555373B1 (en) Methods for identifying human cell lines useful for endogenous gene activation isolated human cell lines identified thereby, and uses thereof
US5827694A (en) DNA encoding non-human animal interferons, vectors and hosts therefor, and recombinant production of IFN polypeptides
EP0167852B1 (de) Menschliches gamma-Interferon
US6455282B1 (en) Cells, vectors and methods for producing biologically active TSH
US5510472A (en) Production of recombinant human interferon-beta2
WO1987005935A1 (en) Methods and compositions for expression of competent eukaryotic gene products
US5646010A (en) Methods and compositions for expression of competent eukaryotic gene products

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE CH DE FR GB IT LI LU NL SE

PUAL Search report despatched

Free format text: ORIGINAL CODE: 0009013

AK Designated contracting states

Kind code of ref document: A3

Designated state(s): AT BE CH DE FR GB IT LI LU NL SE

17P Request for examination filed

Effective date: 19880527

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 19901103

RIN1 Information on inventor provided before grant (corrected)

Inventor name: SARVER, NAVA

Inventor name: DROHAN, WILLIAM N.

Inventor name: RICCA, GEORGE A.